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Sample GSM1440771

Query DataSets for GSM1440771

Status

Public on Jul 28, 2015

Title

PolyA Transcripts replicate 1 (Nitrogen starvation 6 hr)

Sample type

SRA

Source name

PolyA Transcripts (Nitrogen starvation 6 hr)

Organism

Chlamydomonas reinhardtii

Characteristics

strain: 4A+
time: 6 hr

Treatment protocol

N-free (TAP-N) and SO42--free (TAP-S) media were prepared as described (Boyle et al., 2012; Kropat et al., 2011). For N- and S- starvation conditions, cells were grown to mid-log phase, washed twice with depleted media and re-suspended in TAP-N or TAP-S media to a density of 2x10^6 cells/ml (Boyle et al. 2012). Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas (The Journal of biological chemistry 287, 15811-15825; Kropat et al. 2011). A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii (The Plant journal for cell and molecular biology 66, 770-780).

Growth protocol

C. reinhardtii wild type strain 4a+ was cultured using Tris-acetate-phosphate (TAP) medium.

Extracted molecule

polyA RNA

Extraction protocol

Cells were first lysed and total RNA extracted using Trizol and cleaned up using RNeasy column (Qiagen). PolyA+ RNA was isolated from 10ug of total RNA.
RNA was fragmented using RNA Fragmentation Reagents (Ambion). Strand-specific RNA-seq library (Parkhomchuk et al., 2009) was constructed using Illumina DNA Sample Preparation Kit (Illumina) with 10 cycles PCR amplification (Parkhomchuk et al.(2009). Nucleic acids research 37, e123).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

polyAND6hrR1
processed data file: master_gene_model.gtf.gz
processed data file: RNA-Seq_Transcripts_and_Annotation.txt.gz

Data processing

Sequenced reads were trimmed for adaptor sequence and other artifacts. Trimmed reads with average quality ≥20, ≤3 Ns, and ≥32 bases were mapped to C. reinhardtii genome (Phytozome v5.3.1) using TopHat v2.0.8 with Bowtie v2.1.0. Sensitive and fusion mapping was enabled. Valid intron length was 20bp to 25 kbp and minimum distance between intra-chromosomal fusions was 1 Mbp. The library type “fr-firststrand” stated and read alignments with >3 mismatches were discarded.
Cufflinks v2.1.1 (modified to work on compact genomes) was used to reconstruct the transcripts guided with the Phytozome v5.3.1 reference transcriptome (See supplementary file: reference_gene_model.gtf.gz). Multi-mapped read correction was turned on and transcript composed of >50% multi-mapped reads or <25 reads were discarded. The remaining transcript assemblies from different time points were merged into a unified set of gene models, which was then compared to the annotated transcriptome. Potential noise was filtered from these predicted transcripts. Sequential steps of filtering criteria were applied as follows; 1). FPKM >0 for both replicates at least at one time point; 2) length of CDS>=50 amino acid; candidate coding regions were predicted based on transcript sequences using TransDecoder script v2013-02-25 from the Trinity package, 3) Remove “noise” transcripts of CuffDiff classes ‘E’, ‘O’ and ‘P’ and single exon transcripts of CuffDiff classes ‘.’, ‘C’ and ‘I’.
Illumina RTA-1.12 to 1.18 software used for basecalling depending on when library is sequenced.
Genome_build: Chlamydomonas reinhardtii genome (Phytozome v5.3.1); C.reinhardtii_v5.3_genomic_scaffold_plastids.fasta.gz
Supplementary_files_format_and_content: peaks list, tab-delimited text files include FPKM values for each sample and the gene/transcript annotations

Submission date

Jul 21, 2014

Last update date

May 15, 2019

Contact name

Chia-Lin Wei

E-mail(s)

CWei@lbl.gov

Phone

+1 (925) 927-2593

Organization name

DOE Joint Genome Institute